Cell culture and hypoxia
In this study, we used human pancreatic adenocarcinoma cell line BxPC-3 which came from the American Type Culture Collection. The BxPC-3-DKK3 cell line was constructed according to our previous research . RPMI 1640 medium supplemented with 10% fetal bovine serum were used for cells culture (37 °C, 5% CO2). Besides, we used 10 μg/ml mitomycin (MilliporeSigma) pretrested the BxPC-3 cells for 2 h to inhibit expansion and ensure that the appropriate concentration of tumor-to-T cells was maintained. For co-cultivation, CD4+ T cells were first seeded into the lower chamber of a co-cultivation system for adherence, while BxPC-3 or BxPC-3-DKK3 cells were seeded into the upper chamber. After stimulated with 1 μg/ml anti-CD3 antibody (Proteintech, 17,617-1-AP) and 0.5 μg/ml anti-CD28 antibody (eBioscience, 16-0288-81) for 24 h, we started cells collection. Besides, sodium oxamate is the key enzyme of glycolysis which could enhance the hypoxic microenvironment by inhibiting glycolysis. BxPC-3 cells washed by PBS were pretreated with 50 mm sodium oxamate (Abcam) for 24 h and then washed before co-cultivation. Lactic acid (Tokyo Chemical Industry Co., Ltd.) was directly added into the co-culture. We used a three-gas incubator (MiniGalaxy A; RS Biotech) to create Hypoxic conditions (1% O2) by injection of N2 (1% O2/94% N2/5% CO2 atmosphere) at 37 °C.
Peripheral blood mononuclear cell (PBMC) preparation
Blood samples came from donors after the provision of written informed consent. A total of 2 ml heparin-anticoagulated venous blood was diluted and then gently added onto 4 ml Lympholyte®-H Cell Separation Media (Cedarlane®), then ccentrifugated for 20 min (2000 rpm, room temperature). After washed with PBS, we centrifuged three times at 1500 rpm at room temperature again. The pellets were resuspended in 1 ml RMPI 1640 to obtain a PBMC suspension.
Isolation of CD4+ T cells
According to the Isolation Kit (MiltenyiBiotec, Inc.), we isolated the CD4+ T cells. Resuspended 1 × 107 PBMCs in 40 µl PBS (supplemented with 0.5% FBS), labeled with 10 µl CD4+ T-Cell Biotin-Antibody Cocktail for five min at 4 °C, 20 µl CD4+ T-Cell MicroBead Cocktail for 10 min at 4 °C. After centrifuged, we resuspended in 500 µl PBS supplemented with 0.5% FBS. Then separated CD4+ T cells by LS Separation columns and incubated in RMPI 1640 supplemented with 10% FBS, 100 U/ml penicillin, and 100 U/ml streptomycin. Flow cytometric analysis was used to detect the purity of the CD4+ T cells.
Lactic acid detection
Lactic acid was detected by a lactate assay kit (Nanjing Jiancheng Bioengineering Institute, A019-2). After mixed samples with a working solution containing LDH, we then incubated in a 37 °C water bath for 10 min. The optical density (O.D.) values were obtained using the GloMax®-Multi + Detection System (Promega Corporation) at a wavelength of 530 nm. Distilled water was used as the control.
Determination of glucose uptake ability
After washed with glucose-free Krebs–Ringer bicarbonate buffer supplemented with 2% BSA, T cells was then been incubated with glucose-free RMPI 1640 containing 100 μg/ml 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl) amino]-2-deoxyglucose (37 °C, 60 min). Then, digested via trypsinization and followed by immediate assessment of fluorescence intensity using the GloMax®-Multi + Detection System (Promega Corporation) at a wavelength of 485 nm.
CD4+ T cells were seeded and incubated for 96 h prior to harvesting. ATP was measured by ENLITEN ATP Assay System (Promega Corporation).
Detection of cellular viability and proliferation
After incubation for 0, 24, 48, 72, 96, or 120 h, the viability of CD4+ T cells was detected by Cell Counting Kit-8 (CCK-8) assay. O.D. values were determined using the GloMax®-Multi + Detection System (Promega Corporation) at a wavelength of 450 nm.
Detection of apoptosis
After digested via trypsinization, the cells were then counted. Then, 5–10 × 104 resuspended cells were centrifuged (1000×g, 37 °C, five min). The supernatant was aspirated, and 195 μl binding buffer was added to the pellet, stained with 5 μl Annexin V-FITC at 4˚C and then added 5 µl propidium iodide, incubated for another five min under the same conditions. Apoptosis was then evaluated by flow cytometry (BD, USA).
Reverse transcription-quantitative (RT-q) PCR
After isolated from the CD4+ T cells by TRIzol® reagent (Invitrogen, USA), the total RNA was stored in DEPC-treated water at − 80 °C. For usage, DNA was digested by DNase I treatment (37 °C, 30 min). After denatured at 65 °C. for five min, the purified RNA then immediately cooled on ice. After reverse transcribed by RevertAid First Strand cDNA Synthesis Kit, the qPCR was performed by SYBR Green PCR kit on a Step One Plus™ Real-Time PCR System.
The following primers:
CD3: forward,3’-ATGAGCTGTGCACAAAGTGG-5’ and reverse,3’-ACATTGACG GGTTTTTCCTG-5’;
CD28: forward,3’-CAGCAGTACTTGGGTGCTGA-5’ and reverse,3’-TATTTG CCACTGCCATTTCA-5’;
CD25: forward, 3’-AGCGGAGACAGAGGAAGAG-5’ and reverse,3’-GGCAAG CACAACGGATG-5’;
CD69: forward,3’-GTGCTGTAATGAATGTGGTC-5’ and reverse,3’-GTAGCATT TCCTCTGGTAGCC-5’;
CD71: forward,3’-ATGATGGATCAAGCTAGATCAGCAT-5’ and reverse,3’-TTG GTTTTGTGACATTGGCCT-5’;
HLA-DR: forward,3’-GCCTCTTCTCAAGCACTGGGA-5’ and reverse,3’-CCA CCAGACCCACAGTCAGG-5’;
ZAP70: forward,3’-CATGAGTGACTGCTGGATCTACAA-5’ and reverse,3’-GCT GGCCAGGCTGTAGTAACA-5’;
GAPDH: forward,3’-CACATGGCCTCCAAGGAGTAA-5’ and reverse,3’-TGAGG GTCTCTCTCTTCCTCTTGT-5’.
Detection of cellular markers
CD4+ T cells were incubated with human CD3 (cat. no. APC-65060; ProteinTech Group, Inc.), CD28 (cat. no. #62-0289-42; eBioscience; Thermo Fisher Scientific, Inc.), CD25, and CD69 (cat. no. FAB1020A and FAB23591P, respectively; R&D Systems, Inc.), and CD71 and HLA-DR (cat. no. ab9179 and ab20181, respectively; Abcam) antibodies (4˚C). FlowJo software (FlowJo version 7 LLC) was used for data analysis.
Western blot analysis
After concentrated the cell lysates and resolved by SDS-PAGE, the proteins were then transferred to polyvinylidene fluoride membranes and immunoblotted. The density of each band was measured using ImageQuant LAS 500 software (Cytiva). The antibodies included primary antibodies against DKK3 and GAPDH (cat. no. ab126080 and ab181602, respectively; Abcam); active de-phospho-β-catenin, E-cadherin, N-Cadherin, and LDHA (cat. no. #19,807, #14,472, #14,215 and #3582, respectively; Cell Signaling Technology, Inc.); GLUT1 and PDK1 (cat. no. sc-377228 and sc-17765, respectively; Santa Cruz Biotechnology, Inc.); HIF-1α and HK2 (cat. no. AP1029b and AM8606b, respectively; Abgent, Inc.); and secondary horseradish peroxidase-conjugated antibodies (Beyotime Institute of Biotechnology). Western blot analysis of OXPHOS in mitochondria isolated from pancreatic cancer cells was performed using an OXPHOS Monoclonal antibody cocktail (cat. no.# 45–8199, Invitrogen; Thermo Fisher Scientific, Inc.).
The concentrations of multiple cytokines were determined using the ELISA method. The ELISA Kit were purchased from R&D Systems, Inc (RDsystem (Duoset Elisa DY317-05) and RD system (DIF50)). The samples were assessed within 30 min using a DNM-9602 microplate reader (Prolong, Beijing, China), and the data were presented as concentrations (pg/ml).
SPSS 22.0 was used for data analysis. Continuous data that obey the normal distribution were expressed as mean values ± standard deviation; quantitative data that do not follow a normal distribution are represented by the median (interquartile range) [M(P25, P75)]. Comparisons among multiple groups was tested by one-way analysis of variance. A P < 0.05 was considered significant difference.